It is conceivable that the combined existence of multiple AS isoforms and their dynamic change over time, as well as environmental stress, could drive underlying pathophysiological mechanisms that have been overlooked mainly due to technical limitations regarding measurement of cells in the inner ear.
As such, the splicing isoforms in the inner ear remain poorly characterized. The current mouse genome annotations are primarily based on the results of short-read RNA sequencing from nonauditory tissues 5, 6.
It is estimated that more than 60% of multiexonic genes in mice undergo AS, which has the potential to create multiple functional protein isoforms from a single gene locus 2. Alternative splicing (AS) plays an instrumental role in cell differentiation, development 3, tissue-identity acquisition, and maintenance of cell-type-specific properties 1, 4, 5. Mammalian genes typically produce multiple transcripts 1, 2, contributing to their transcriptomic and proteomic diversity relative to other organisms. RNA expression at the isoform level is biologically more informative than at the gene level, and different isoforms of the same gene can have different biological functions. This work expands our mechanistic understanding of auditory functions at the level of isoform resolution. Furthermore, we found that otoferlin isoforms are associated with IHC functions and auditory phenotypes. Upon deletion of the canonical otoferlin isoform, the identified short isoform is able to support normal hearing thresholds but with reduced sustained exocytosis of IHCs, and further revealed otoferlin functions in endocytic membrane retrieval that was not well-addressed previously. To illustrate the importance of identifying spliced isoforms, we investigated otoferlin, a key protein involved in synaptic transmission in inner hair cells (IHCs). Then we analyzed and verified the unannotated transcripts using RNA-seq, RT-PCR, Sanger sequencing, and MS-based proteomics approaches. We integrated single-cell short-read and long-read RNA sequencing techniques and identified 236,012 transcripts, 126,612 of which were unannotated in the GENCODE database.
In the cochlea, the repertoire of alternative isoforms remains unexplored. Isoforms of a gene may contribute to diverse biological functions.
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